Solexa sequencing and custom microRNA chip reveal repertoire of microRNAs in mammary gland of bovine suffering from natural infectious mastitis.

Identification of microRNAs (miRNAs), target genes and regulatory networks associated with innate immune and inflammatory responses and tissue damage is essential to elucidate the molecular and genetic mechanisms for resistance to mastitis. In this study, a combination of Solexa sequencing and custom miRNA chip approaches was used to profile the expression of miRNAs in bovine mammary gland at the late stage of natural infection with Staphylococcus aureus, a widespread mastitis pathogen. We found 383 loci corresponding to 277 known and 49 putative novel miRNAs, two potential mitrons and 266 differentially expressed miRNAs in the healthy and mastitic cows' mammary glands. Several interaction networks and regulators involved in mastitis susceptibility, such as ALCAM, COL1A1, APOP4, ITIH4, CRP and fibrinogen alpha (FGA), were highlighted. Significant down-regulation and location of bta-miR-26a, which targets FGA in the mastitic mammary glands, were validated using quantitative real-time PCR, in situ hybridization and dual-luciferase reporter assays. We propose that the observed miRNA variations in mammary glands of mastitic cows are related to the maintenance of immune and defense responses, cell proliferation and apoptosis, and tissue injury and healing during the late stage of infection. Furthermore, the effect of bta-miR-26a in mastitis, mediated at least in part by enhancing FGA expression, involves host defense, inflammation and tissue damage.

Glucagon-like peptide-1(GLP-1) receptor agonists: potential to reduce fracture risk in diabetic patients?

This review summarizes current knowledge about glucagon-like peptide 1 receptor agonists (GLP-1 RA) and their effects on bone metabolism and fracture risk. Recent in vivo and in vitro experiments indicated that GLP-1 RA could improve bone metabolism. GLP-1 could affect the fat-bone axis by promoting osteogenic differentiation and inhibiting adipogenic differentiation of bone mesenchymal precursor cells (BMSCs), which express the GLP-1 receptor. GLP-1 RA may also influence the balance between osteoclasts and osteoblasts, thus leading to more bone formation and less bone resorption. Wnt/ß-catenin signalling is involved in this process. Mature osteocytes, which also express the GLP-1 receptor, produce sclerostin which inhibits Wnt/ß-catenin signalling by binding to low density lipoprotein receptor-related protein (LRP) 5 and preventing the binding of Wnt. GLP-1 RA also decreases the expression of sclerostin (SOST) and circulating levels of SOST. In addition, GLP-1 receptors are expressed in thyroid C cells, where GLP-1 induces calcitonin release and thus indirectly inhibits bone resorption. Furthermore, GLP-1 RA influences the osteoprotegerin(OPG)/receptor activator of nuclear factor-κB ligand (RANKL)/receptor activator of nuclear factor-κB (RANK) system by increasing OPG gene expression, and thus reverses the decreased bone mass in rats models. However, a recent meta-analysis and a cohort study did not show a significant relationship between GLP-RA use and fracture risk. Future clinical trials will be necessary to investigate thoroughly the relationship between GLP-1 RA use and fracture risk in diabetic patients.

iTRAQ-proteomics and bioinformatics analyses of mammary tissue from cows with clinical mastitis due to natural infection with Staphylococci aureus.

BACKGROUND: Proteomics and bioinformatics may help us better understand the biological adaptations occurring during bovine mastitis. This systems approach also could help identify biomarkers for monitoring clinical and subclinical mastitis. The aim of the present study was to use isobaric tags for relative and absolute quantification (iTRAQ) to screen potential proteins associated with mastitis at late infectious stage. RESULTS: Healthy and mastitic cows' mammary gland tissues were analyzed using iTRAQ combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS). Bioinformatics analyses of differentially expressed proteins were performed by means of Gene Ontology, metabolic pathways, transcriptional regulation networks using Blast2GO software, the Dynamic Impact Approach and Ingenuity Pathway Analysis. At a false discovery rate of 5%, a total of 768 proteins were identified from 6,499 peptides, which were matched with 15,879 spectra. Compared with healthy mammary gland tissue, 36 proteins were significantly up-regulated (>1.5-fold) while 19 were significantly down-regulated (<0.67-fold) in response to mastitis due to natural infections with Staphylococci aureus. Up-regulation of collagen, type I, alpha 1 (COL1A1) and inter-alpha (Globulin) inhibitor H4 (ITIH4) in the mastitis-infected tissue was confirmed by Western blotting and Immunohistochemistry. CONCLUSION: This paper is the first to show the protein expression in the late response to a mastitic pathogen, thus, revealing mechanisms associated with host tissue damage. The bioinformatics analyses highlighted the effects of mastitis on proteins such as collagen, fibrinogen, fibronectin, casein alpha and heparan sulfate proteoglycan 2. Our findings provide additional clues for further studies of candidate genes for mastitis susceptibility. The up-regulated expression of COL1A1 and ITIH4 in the mastitic mammary gland may be associated with tissue damage and repair during late stages of infection.

Pyogenic arthritis, pyoderma gangrenosum, and acne syndrome in a Chinese family: A case report and review of literature.

BACKGROUND: Pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome is a rare autosomal dominant genetic disease characterized by severe autoimmune inflammation, caused by mutations in the PSTPIP1 gene. Due to PAPA heterogeneous clinical manifestation, misdiagnosis or delayed diagnoses are difficult to avoid. With the use of whole-exome sequencing, we identified a missense mutation in the PSTPIP1 gene in a Chinese family. To the best of our knowledge, this is the first case of PAPA reported in China. CASE SUMMARY: A 9-year-old boy suffered from recurrent aseptic pyogenic arthritis triggered by minor trauma or few obvious predisposing causes for more than 3 years. Pyogenic arthritis occurred every 3-5 mo, affecting his knees, elbows, and ankle joints. Treatments, such as glucocorticoids, antibiotics, even surgeries could alleviate joints pain and swelling to some extent but could not inhibit the recurrence of arthritis. Similar symptoms were present in his younger brother but not in his parents. According to the whole-exome sequencing, a missense mutation in exon 11 of the PSTPIP1 gene (c.748G>C; p.E250Q) was detected in the boy, his younger brother and his father. Taking into account the similar phenotypic features with PAPA syndrome reported previously, we confirmed a diagnosis of PAPA syndrome for the family. CONCLUSION: In this case, a missense mutation (c.748G>C; p.E250Q) in PSTPIP1 gene was identified in a Chinese family with PAPA syndrome. Previous studies emphasize the fact that PAPA syndrome is hard to diagnose just through the clinical manifestations owing to its heterogeneous expression. Genetic testing is an effectual auxiliary diagnostic method, especially in the early stages of pyogenic arthritis. Only if we have a deep understanding and rich experience of this rare disease can we make a prompt diagnosis, develop the best clinical treatment plan, and give good fertility guidance.

Cellular Communication in Bone Homeostasis and the Related Anti-osteoporotic Drug Development.

Background：Intercellular crosstalk among osteoblast, osteoclast, osteocyte and chondrocyte is involved in the precise control of bone homeostasis. Disruption of this cellular and molecular signaling would lead to metabolic bone diseases such as osteoporosis. Currently a number of anti-osteoporosis interventions are restricted by side effects, complications and long-term intolerance. This review aims to summarize the bone cellular communication involved in bone remodeling and its usage to develop new drugs for osteoporosis. Methods：We searched PubMed for publications from 1 January 1980 to 1 January 2018 to identify relevant and latest literatures, evaluation and prospect of osteoporosis medication were summarized. Detailed search terms were 'osteoporosis', 'osteocyte', 'osteoblast', 'osteoclast', 'bone remodeling', 'chondrocyte', 'osteoporosis treatment', 'osteoporosis therapy', 'bisphosphonates', 'denosumab', 'Selective Estrogen Receptor Modulator (SERM)', 'PTH', 'romosozumab', 'dkk-1 antagonist', 'strontium ranelate'. Results：A total of 170 papers were included in the review. About 80 papers described bone cell interactions involved in bone remodeling. The remaining papers were focused on the novel advanced and new horizons in osteoporosis therapies. Conclusion：There exists a complex signal network among bone cells involved in bone remodeling. The disorder of cell-cell communications may be the underlying mechanism of osteoporosis. Current anti-osteoporosis therapies are effective but accompanied by certain drawbacks simultaneously. Restoring the abnormal signal network and individualized therapy are critical for ideal drug development.

Liraglutide and Insulin Have Contrary Effects on Adipogenesis of Human Adipose-Derived Stem Cells via Wnt Pathway.

BACKGROUND: Glucagon-like peptide-1 (GLP-1) has been reported to have beneficial impacts on improving human's metabolism and ameliorating insulin resistance. While insulin is another important and conventional drug in diabetes treatment, but it has an adverse effect on weight gain. PURPOSE: To make sure whether GLP-1 and insulin play different roles in human adipose-derived stem cells (hADSCs). METHODS: We examined the in vitro roles and molecular mechanisms of liraglutide, a GLP-1 analogue, and human insulin on hADSCs isolated from subcutaneous adipose tissue. Different concentrations (0, 0.1, 1, 10, 100nM) of liraglutide and insulin were added to proliferation and differentiation medium of hADSCs, respectively. RESULTS: Liraglutide inhibits while insulin promotes the proliferation and differentiation at the concentration of 100nM. Moreover, the levels of GSK-3 increase during differentiation and liraglutide could down-regulate it when compared with insulin. We also find that the activation of phosphorylated GSK-3α and GSK-3ß is involved in the differentiation roles. And classical and non-classical Wnt pathways all play roles in the differentiation, which are characterized with the up/down-regulation of the expression of adipogenesis genes such as PPAR-γ and CEBP-α. CONCLUSION: Liraglutide and insulin have contrary effects on the proliferation and adipogenesis via Wnt pathway in primary cultured ADSCs. Those effects could partly explain the different roles of GLP-1 and insulin on weight gain and insulin resistance.

Lung Cancer Cells Derived Circulating miR-21 Promotes Differentiation of Monocytes into Osteoclasts.

OBJECTIVE: Osteoclastogenesis is a key process in osteolytic bone metastasis (BM). Previous studies indicated that some miRNAs could regulate cancers progression and osteoclastogenesis. Our purpose was to investigate the roles of lung cancer cells-derived circulating miR-21 on osteoclastogenesis and its clinical significance in BM patients. MATERIALS AND METHODS: The difference of miRNA expression in two lung cancer cell lines SBC-5 (with characteristic BM ability) and SBC-3 (without BM ability) were analyzed by microarray and qRT-PCR. Circulating miR-21 levels of lung cancer patients with or without BM were compared by qRT-PCR. The TRAP staining was used to investigate the effects of conditioned media from lung cancer cell lines or patients' plasma with different miR-21 levels on osteoclastogenesis. ROC curve was used to evaluate the diagnostic performance of circulating miR-21 in BM patients. RESULTS: We found that miR-21 expression was specifically higher in SBC-5 than that in SBC-3 cells. The supernatants of SBC-5 cells with higher-level miR-21 promoted osteoclastogenesis. Moreover, we demonstrated that the circulating miR-21 level was significantly higher in BM patients than that in non-BM patients. The plasma from BM patients with higher-level miR-21 could also promote osteoclastogenesis. Mechanistically, lung cancer cells-derived circulating miR-21 could be transferred into osteoclast precursor cells and promote osteoclastogenesis probably by inhibiting PTEN. Finally, clinical data showed that circulating miR-21 had a potential for the diagnosis of BM. CONCLUSION: Overall, our findings suggested that circulating miR-21 played important roles in osteoclastogenesis of lung cancer patients and may serve as a biomarker to diagnose BM of lung cancer.

Bone marrow adipocytes enhance osteolytic bone destruction by activating 1q21.3(S100A7/8/9-IL6R)-TLR4 pathway in lung cancer.

PURPOSE: Bone metastasis is the result of complex crosstalk between tumor cells and bone marrow cells. Bone marrow adipocytes (BMAs) are the most abundant cell type in adult bone marrow. Therefore, we explore the effects of BMAs on bone metastasis in lung cancer. METHODS: RNA-seq was used to compare the mRNA expression level of bone metastatic SBC5 cells and non-bone metastatic SBC3 cells. Rosiglitazone-induced marrow adiposity and intra-femoral injection of SBC5 cells were used to demonstrate the relationship between BMAs and SBC5 cells in vivo. Co-culture system, gene co-expression, gene ontology (GO) enrichment analysis and protein-protein interaction (PPI) network were used to explore the potential mechanism. RESULTS: BMAs specially enhance the invasion of bone metastatic SBC5 instead of non-bone metastatic SBC3 in vitro. SBC5 instead of SBC3 promoted osteoblast and osteoclast differentiation as well as de-differentiation of mature BMAs. Rosiglitazone-induced marrow adiposity significantly enhanced osteolytic lesion induced by SBC5 in vivo. RNA-seq revealed that compared with SBC3, S100A9 and S100A8 genes were the most prominent genes up-regulated in SBC5 cells. High expression of S100A8/9 in SBC5 could be responsible for the crosstalk between lung cancer cells and BMAs. More importantly, interleukin 6 receptor (IL6R), which is adjacent to S100A8/A9 in 1q21.3, was significantly up-regulated by BMAs in vitro. S100A8/A9 (1 µg/ml) could obviously enhance the osteoblastic differentiation and inhibit adipogenic differentiation, whereas TLR4 inhibitor TAK242 (10 µmol/l) significantly attenuated this effect. CONCLUSIONS: Our study suggested that bone marrow adipocyte may communicate with lung cancer cells via 1q21.3 (S100A8/A9-IL6R)-TLR4 pathway to promote osteolytic bone destruction. 1q21.3 (S100A8/A9-IL6R) is a potential target for the treatment of lung cancer bone metastasis.

A novel compound mutation in alpha-L-iduronidase gene causes mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPSI) is a rare autosomal recessive disorder caused by mutations in alpha-L-iduronidase (IDUA) gene. IDUA contributes to the degradation of the glycosaminoglycans, including heparan sulphate and dermatan sulphate. Deficient activity of IDUA generates accumulation of glycosaminoglycans in lysosomes leading to MPS I. Here, we identified two boys with MPS I caused by a compound heterozygote of a reported c.265C > T (p.R89W) missense mutation in exon 2 and a novel c.1633G > T (p.E545*, 109) nonsense mutation in exon 11 of IDUA gene in a Chinese family. R89 is close to the active site and its replacement will affect the structure and function of IDUA. Besides, termination from E545 deletes one of the prominent domains and alters the spatial structure of IDUA. In conclusion, our study demonstrates a previously unrecognized mutation in IDUA gene and this report adds to the mutational spectrum observed.

Bone Marrow Adipocyte: An Intimate Partner With Tumor Cells in Bone Metastasis.

The high incidences of bone metastasis in patients with breast cancer, prostate cancer and lung cancer still remains a puzzling issue. The "seeds and soil" hypothesis suggested that bone marrow (soil) may provide a favorable "niche" for tumor cells (seed). When seeking for effective ways to prevent and treat tumor bone metastasis, most researchers focus on tumor cells (seed) but not the bone marrow microenvironment (soil). In reality, only a fraction of circulating tumor cells (CTCs) could survive and colonize in bone. Thus, the bone marrow microenvironment could ultimately determine the fate of tumor cells that have migrated to bone. Bone marrow adipocytes (BMAs) are abundant in the bone marrow microenvironment. Mounting evidence suggests that BMAs may play a dominant role in bone metastasis. BMAs could directly provide energy for tumor cells, enhance the tumor cell proliferation, and resistance to chemotherapy and radiotherapy. BMAs are also known for releasing some inflammatory factors and adipocytokines to promote or inhibit bone metastasis. In this review, we made a comprehensive summary for the interaction between BMAs and bone metastasis. More importantly, we discussed the potentially promising methods for the prevention and treatment of bone metastasis. Genetic disruption and pharmaceutical inhibition may be effective in inhibiting the formation and pro-tumor functions of BMAs.

Interferon-Gamma-Mediated Osteoimmunology.

Osteoimmunology is the interdiscipline that focuses on the relationship between the skeletal and immune systems. They are interconnected by shared signal pathways and cytokines. Interferon-gamma (IFN-γ) plays important roles in immune responses and bone metabolism. IFN-γ enhances macrophage activation and antigen presentation. It regulates antiviral and antibacterial immunity as well as signal transduction. IFN-γ can promote osteoblast differentiation and inhibit bone marrow adipocyte formation. IFN-γ plays dual role in osteoclasts depending on its stage. Furthermore, IFN-γ is an important pathogenetic factor in some immune-mediated bone diseases including rheumatoid arthritis, postmenopausal osteoporosis, and acquired immunodeficiency syndrome. This review will discuss the contradictory findings of IFN-γ in osteoimmunology and its clinical application potential.

Ovariectomy-induced bone loss in TNFα and IL6 gene knockout mice is regulated by different mechanisms.

We examined the effects of tumor necrosis factor-α (TNFα) and interleukin-6 (IL6) gene knockout in preserving the bone loss induced by ovariectomy (OVX) and the mechanisms involved in bone metabolism. Twenty female wild-type (WT), TNFα-knockout (TNFα-/-) or IL6-knockout (IL6-/-) mice aged 12 weeks were sham-operated (SHAM) or subjected to OVX and killed after 4 weeks. Bone mass and skeletal microarchitecture were determined using micro-CT. Bone marrow stromal cells (BMSCs) from all three groups (WT, TNFα-/- and IL6-/-) were induced to differentiate into osteoblasts or osteoclasts and treated with 17-ß-estradiol. Bone metabolism was assessed by histological analysis, serum analyses and qRT-PCR. OVX successfully induced a high turnover in all mice, but a repair effect was observed in TNFα-/- and IL6-/- mice. The ratio of femoral trabecular bone volume to tissue volume, trabecular number and trabecular thickness were significantly decreased in WT mice subjected to OVX, but increased in TNFα-/- mice (1.62, 1.34, 0.27-fold respectively; P < 0.01) and IL6-/- mice (1.34, 0.80, 0.22-fold respectively; P < 0.01). Furthermore, we observed a 29.6% increase in the trabecular number in TNFα-/- mice when compared to the IL6-/- mice. Both, TNFα-/- and IL6-/- BMSCs exhibited decreased numbers of TRAP-positive cells and an increase in ALP-positive cells, with or without E2 treatment (P < 0.05). While the knockout of TNFα or IL6 significantly upregulated mRNA expressions of osteoblast-related genes (Runx2 and Col1a1) and downregulated osteoclast-related mRNA for TRAP, MMP9 and CTSK in vivo and in vitro, TNFα knockout appeared to have roles beyond IL6 knockout in upregulating Col1a1 mRNA expression and downregulating mRNA expressions of WNT-related genes (DKK1 and Sost) and TNF-related activation-induced genes (TRAF6). TNFα seemed to be more potentially invasive in inhibiting bone formation and enhancing TRAF6-mediated osteoclastogenesis than IL6, implying that the regulatory mechanisms of TNFα and IL6 in bone metabolism may be different.

Immune Cells Act as Promising Targets for the Treatment of Bone Metastasis.

BACKGROUND: Bone metastasis is a common complication of certain types of cancer, and unfortunately, it is still incurable to date. Immune system is a major defence against tumor cells and bone metastasis. However, the immunodeficient mouse models are widely used in most researches of bone metastasis, thereby excluding the regulatory roles of the immune system in bone metastasis. OBJECTIVE: The aim was to provide a comprehensive overview on the cellular and molecular mechanisms by which immune cells interact with tumor cells and bone cells, as well as recent related patents. METHOD: We performed a literature search of PubMed database for the current knowledge on the interaction between bone metastasis and immune system. Recent related patents were obtained from World Intellectual Property Organization (WIPO) website. RESULTS: Immune cells in the bone-tumor microenvironment include dentritic cells, monocytes/ macrophages, myeloid-derived suppressor cells, regulatory T cells, T helper cells, neutrophil, CD4+ T lymphocytes, CD8+cytotoxic T lymphocytes, and natural killer cells, et al. In addition to their well-known immunomodulatory function, recent studies revealed that immune cells could cooperate with tumor cells and bone cells to enhance bone metastasis and tumor progression. There are some patents targeting immune system to inhibit bone metastasis. CONCLUSION: Immune cells are instigated by tumor cells to enhance the occurrence and the development of bone metastasis. Taking full advantage of anti-tumor roles of immune cells may bring the promise of a possible cure for bone metastasis.

Genetic mutations potentially cause two novel NCF1 splice variants up-regulated in the mammary gland, blood and neutrophil of cows infected by Escherichia coli.

Neutrophil cytosolic factor 1 (NCF1) plays a crucial role in host defense against microbial pathogens. In this study, we investigated the potential alternative splicing patterns, expression and splice-relevant single nucleotide polymorphisms (SNPs) of the bovine NCF1 gene to increase insights into its potential role against bovine mastitis caused by Escherichia coli infection. Using RT-PCR and clone sequencing methods, we found two novel splice variants designed as NCF1-TV1 (retained intron 6) and NCF1-TV2 (retained part of intron 8), respectively, encoding two putative truncated proteins (239AA and 283AA). Two splice variants were drastically up-regulated in the mastitis-infected cows' mammary tissues, blood and neutrophils compared with these of healthy cows using real-time RT-PCR. Genomic sequencing analysis identified four novel SNPs g.10112 G>A, g.10766 T>C, SNPs g.12085 G>A and g.12430 T>C at the ends of intron 6 and intron 8 of NCF1. ESE motif predicted that the SNP (g.10766 T>C) might affect the binding with splicing-related factors and subsequently caused the production of aberrant splice variant NCF1-TV1, which is one of the potential reasons that the functional SNP was associated with increased milk somatic cell score in cow. Our results would help in better understanding the NCF1 gene function in the process against pathogen infection, and the effect of splicing-related SNP on the production of aberrant splice variant and careful functional characterization in dairy cattle.

Fasting hyperglycemia increases in-hospital mortality risk in nondiabetic female patients with acute myocardial infarction: a retrospective study.

Previous studies had shown that elevated admission plasma glucose (APG) could increase mortality rate and serious complications of acute myocardial infarction (AMI), but whether fasting plasma glucose (FPG) had the same role remains controversial. In this retrospective study, 253 cases of AMI patients were divided into diabetic (n = 87) and nondiabetic group (n = 166). Our results showed that: compared with the nondiabetic patients, diabetic patients had higher APG, FPG, higher plasma triglyceride, higher rates of painless AMI (P < 0.01), non-ST-segment elevation myocardial infarction (NSTEMI), and reinfraction (P < 0.05). They also had lower high density lipoprotein cholesterol and rate of malignant arrhythmia, but in-hospital mortality rate did not differ significantly (P > 0.05). While nondiabetic patients were subgrouped in terms of APG and FPG (cut points were 11.1 mmol/L and 7.0 mmol/L, resp.), the mortality rate had significant difference (P < 0.01), whereas glucose level lost significance in diabetic group. Multivariate logistic regression analysis showed that FPG (OR: 2.014; 95% confidence interval: 1.296-3.131; p < 0.01) but not APG was independent predictor of in-hospital mortality for nondiabetic patients. These results indicate that FPG can be an independent predictor for mortality in nondiabetic female patients with AMI.